<p>Lysosomal two-pore channels (TPC) trigger Ca²⁺ release from the endoplasmic reticulum (ER). The ensuing ER Ca²⁺ depletion activates STIM1-gated store-operated Ca²⁺ entry (SOCE) channels that sustain Ca²⁺ signals regulating fundamental cellular processes. How TPC channels and STIM1 integrate distinct intra and extracellular cues is unclear. Here, we show that TPC2 activation inhibits SOCE by enforcing rapid and persistent Ca²⁺-CaM-dependent inactivation of the STIM-Orai activating region (SOAR). The TPC2 activators NAADP and TPC2-A1-N abrogated SOCE in multiple cell lines and enhanced the slow Ca²⁺ dependent inactivation (SCDI) of STIM1-gated Orai1 channels. TPC2 engagement triggered lysosomal Ca²⁺ release and mobilized ER Ca²⁺ stores but prevented RFP-STIM1 recruitment to the TIRF plane by thapsigargin and disassembled RFP-STIM1 clusters forming after store depletion, preventing and acutely reversing SOCE. These effects persisted in STIM1 mutants truncated after the SOAR and were prevented by TPC2 genetic or pharmacological invalidation, Calmodulin (CaM) inhibition, and cytosolic Ca²⁺ chelation. We conclude that Ca²⁺ ions released by TPC2 channels on lysosomes regulate CaM-dependent STIM1 inactivation.</p>